Transferrin glycans composition for the induction of immune tolerance

ABSTRACT

The invention pertains to a process for the production of a pharmaceutical composition effective for controlling in a recipient mammalian host, particularly man, immune reactions of the type that are involved in graft of foreign tissue or cells, particularly transplantation of foreign tissues, organs or cells, particularly of allogeneic or even xenogeneic origin, or in immunodeficiency-linked diseases, which pharmaceutical composition is characterized by an active principle consisting of pooled transferrin-derived glycans obtained from a number of donors sufficient to allow the pool to contain sufficient phenotypic information required to ensure an induction of tolerance against antigens in an immuno-depressed host grafted with said antigens, after that host had been administered an amount of such pooled transferrin glycans effective to induce said tolerance.

This application is a national stage application under 35 U.S.C. §371 ofPCT/EP98/03159. The contents of which are hereby incorporated byreference.

FIELD OF INVENTION

The invention relates to the production of compositions, particularlypharmaceutical compositions, containing one or several active principlescapable of controlling the immune reactions of a host against allogeneicor xenogeneic cells, tissues or organs or of immuno-competent cellsagainst an immuno-incompetent or immuno-suppressed host, particularlythose immune reactions which are involved in the so-called hostversus-graft reaction (HvGR) and so-called graft versus-host reaction(GvHR) or graft versus-host disease (GvHD), as well as immune reactionswhich are brought into play in bone marrow transplantation (BMT), i.e.when the host is transplanted with allogeneic or xenogeneic incompatiblebone marrow.

DESCRIPTION OF THE RELATED ART

Series of studies have been initiated in 1978 by one of the inventorsrelative to the bone-marrow-engraftment-promoting activity ofbone-marrow-derived factors (Pierpaoli W. et al, Transplantation 1978;26: 456-458) and (Pierpaoli W. et al, J. Clin. and Lab. Immunol. 1985;16:115-124). The initial observation was that the supernatant of asolution in which the bone marrow cells had been suspended (bone marrowsupernatant: BM-SN) provided an engraftment-enhancing activity(Pierpaoli W. et al, Cell Immunol 1980; 52:62-72). This indicated thepresence of factors able to modify the capacity of the bone marrow to beengrafted in an irradiated host for induction of permanent allogeneic orxenogeneic chimerism.

An extensive series of experiments further demonstrated thathigh-molecular-weight fractions obtained by ultrafiltration throughporous membranes of the native BM-SN derived from rabbit marrowcontained marrow-regulating factors (MRF) capable of exerting the sameeffect, i.e., of inducing hemopoietic chimerism across the H-2 barrierin the murine model (Pierpaoli W. et al, Cell Immunol 1981; 57:219-228).However, the results obtained were not easily reproduced, at leastquantitatively; there was considerable variability in the results andthe incidence of secondary disease was high. Moreover, induction ofchimerism was not achieved in all of the murine H-2 combinations tested(Pierpaoli W. et al, J Lab Clin Immunol 1985; 16:115-124).

European Patent application n° EP89403103.8/0426924 Pierpaoli W. et al,Cell Immunol 1981;57:219-228 and Pierpaoli W. et al, J Lab Clin Immunol1985;16:115-124) reported that a specific component from rabbitbone-marrow-derived fractions, namely transferrin, could be responsiblefor the facilitation of allogeneic and xenogeneic bone marrowengraftment that had been achieved earlier with rabbit and bovine,marrow-derived fractions. Treating lethally irradiated C57BL/6 micetransplanted with bone marrow from BALB/c donors with iron-saturatedhuman transferrin or conalbumin, resulted in remarkably stableengraftment, avoidance of GvHD and enduring chimerism in the majority oftest animals (Pierpaoli W. et al, Cell Immunol 1991;134:225-234).

Transferrins as such have been abundantly studied. They consist oftwo-sited, single-chain proteins capable of binding metals. They arewidely distributed in physiological fluids and cells of vertebrates.

Each of the transferrin molecules consists of a single polypeptidechain, of molecular weight in the range 76,000-81,000, which containstwo similar but not identical iron binding sites. Human serumtransferrin contains about 5% carbohydrate, linked to the protein in twoidentical and nearly symmetrical branched heterosaccharide chains. Ithas a molecular weight of about 80,000. 1 mg of the iron-saturatedprotein contains about 1.4 μg iron.

The complete amino acid sequence of human plasma transferrin hasrecently been established by at least three groups using CNBr cleavage(CNBr) and by complementary DNA (cDNA) methods (MacGillivray, R. T. A.,et al. “The complete amino acid sequence of human serum transferrin”.Proc. Natl. Acad. Sci. USA 79: 2504-2508, 1982 and Uzan, G. et al.“Molecular cloning and sequence analysis of cDNA for human transferrin.Biochem. Biophys. res. Commun. 119: 273-281, 1984 and Yang, F. et al.“Human transferrin: cDNA characterization and chromosomal localization”.Proc. Natl. Acad. Sci. USA 81: 2752-2756, 1984). It is composed of 678amino acid residues, which together with the two-N-linkedoligosaccharide chains exhibit a calculated molecular weight of 79,570(of which 6% is contributed by the glucosidic moiety: MacGillivray, R.T. A. et al. and Uzan G. et al., “Molecular cloning and sequenceanalysis of cDNA for human transferrin”. Biochem. Biophys. Res. Commun.119:273-281, 1984). Wiliams J. (“The evolution of transferrin”, TrendsBiochem. Sci, 7: 394-397, 1982) has suggested the importance ofsulfhydryl groups in stabilizing the iron-binding site and has tracedtheir evolutionary development to the 17 disulfides found in humantransferrin.

For a general review of the status of general knowledge abouttransferrins see the general publication titled “The Physiology ofTransferrin and Transferrin Receptors” by Helmut A. Huebbers and ClementA. Finch in Physiological Reviews, vol. 67, No. 2, April 1987. Thatpublication discloses also procedures for obtaining transferrin.Particularly transferrin of human origin in a biologically pure statehas been disclosed in that publication. Preferred purificationprocedures are based on physicochemical separation steps followed byselective fixation on matrix-bound antibody or matrix-bound receptor.

Purified iron-saturated transferrin in a substantially biologically purestate, is substantially free of serum albumin proteins.

While the capability referred to above of iron-saturated humantransferrin to protect lethally irradiated C5713L/6 mice recipientstransplanted with bone marrow from BALB/c mice donors has beendemonstrated, the engraftment-promoting activity of human transferrin inother H2-incompatible murine combinations was not as successful in allinstances.

Human transferrin induced no immune tolerance in C57BL/6 mice graftedwith marrow from C3H/He donors. Further work let the inventor to thenconsider that the promoting effects of transferrin rather variedaccording to the histogenetic H-2-type combination used, the promotingeffect being maximal in C57BL/6 mice (H-^(2b)) grafted with BALB/c(H-2^(d)) marrow and absent in C57BL/6 mice grafted with C3H/HE(H-2^(k)) marrow (Pierpaoli W. Nat. Immun. 1992; 11:356-365).

Thus it appears that the capability of plasma-derived transferrins (Tf)to profoundly affect engraftment of allogeneic or xenogeneic bone marrowin lethally irradiated mice and to produce a lasting chimerism, dependson a matching at least to some degree of the donor's transferrins andthe cell and tissue antigens in the immunosuppressed and transplantedhost. Indeed an induction of a durable state of immunologicalunresponsiveness or “tolerance” and, accordingly, a facilitation ofengraftment of donor xenogeneic or allogeneic antigens, e.g., bonemarrow, in irradiated or chemically immunosuppressed recipients treatedwith transferrin of a same donor is obtained, when said recipients areadministered with the donor's transferrins and antigens, simultaneouslyor sequentially. A properly timed presentation of both transferrin andantigens, e.g., human transferrin and human leukocytes inimmunosuppressed mice during initial recovery of their immune tissuesand cells, results later in their inability to “recognize” human donorlymphocytes and to mount an immediate- or a delayed-type immune responseagainst the antigens of the corresponding human donor. This “tolerant”state of the recipient mice has been confirmed by the absence ofcytotoxic antibodies in the “tolerant” mouse sera against the humandonor lymphocytes, by the inability of “tolerant” mice to mount acell-mediated immune reaction in vivo against human donor leukocytes,and finally by an in vitro lack of reactivity of splenocytes of the“tolerant” mouse towards irradiated human donor lymphocytes. In summary,a donor's blood plasma-derived transferrin have the remarkable capacityof inducing a state of durable unresponsiveness in an immunosuppressedrecipient host administered with antigens of the same donor in thecourse of regeneration of stem cells and immunocompetent cells in itsbone. marrow and lymphatic organs.

It would thus seem that transferrin is a major element of theself-recognition and immune mechanisms and that it participates to thedevelopment and maintenance of self-tolerance during ontogeny andadulthood. That could possibly account at least in part for the geneticpolymorphism and heterogeneity of human serum transferrins possiblymimicking the “immune personality” of a human individual: see thearticle titled “The Biology of transferrin” of de Jong G., Dijk J. P.and Van Ejk, H. C. Clinica Chimica ACTA, 1990, 1-46, Vol. 190.

The latter Clinica Chimica Acta publication should also be used as areference for the definition of transferrins as used herein. Thisexpression is to be interpreted broadly. Transferrins are deemed toconsist of all molecules which, as indeed provided by that article, areincluded in that class of compounds designated as a whole astransferrin. They include the so-called apo-transferrins, saturatedtransferrin, ferro-transferrins, etc.

While the above-mentioned method appears suitable to produce a safe,specific and rapid condition in a recipient host for rapid engraftmentof histoincompatible cells or tissues of a donor host and even isamenable to be adapted to different species, and also to man, in a largevariety of pathologies, its use will not be all too easy under manypractical circumstances, particularly where such method would alwaysnecessarily involve an identity between the donor of the transferrinsand the donor of the antigens to be transplanted in the recipient host.

The finding that as efficient an induction of immune tolerance in arecipient host with respect to antigens, e.g., bone marrow or organs ofallogeneic or xenogeneic nature, can also be achieved upon usingmixtures of transferrins obtained from a limited number of donors of thesame species as that of the antigens to be transplanted (e.g., humanswhen the particular donor is a human, piglets when the donor is apiglet, etc.) was thus all the more remarkable. For instance aninduction of immune tolerance against antigens of a human donor can beachieved in mice by a treatment thereof with human transferrins obtainedfrom plasma mixtures resulting from the pooling of plasmas obtained froma fairly limited number of individuals. In other words it appeared thattransferrins obtained from pooled plasmas—as they are used in theindustry concerned with the extraction of determined blood factors fromsuch pooled plasmas—contained enough of the phenotypic informationrequired to ensure in mice, a specific tolerance against the antigens ofmost, let alone all human donors.

Hence mixtures or “pools” of transferrins (hereafter referred to as“pooled transferrin” or p-Tf) can be obtained from a limited number ofdonors, yet sufficient to allow said mixtures or pools to contain enoughof the phenotypic information required to ensure an induction oftolerance in a given immuno-depressed recipient host grafted withantigens of a given donor host, after that recipient host had beenadministered an amount of such pooled transferrins.

It thus appears that the cumulated phenotypic information collected fromthe individuals who provided the pooled transferrins is sufficient toinduce a specific immune tolerance in different recipient hosts of thesame species, as if the pooled transferrins had also contained thetransferrins of the individual donor of the antigens to be grafted, nomatter whether that individual was among the group of individuals whoseplasmas—or transferrins—were pooled, or not.

Advantageously, pooled transferrins are obtained from human plasma poolsproduced in the industry of blood products. Such plasma pools oftenoriginate from several hundreds to several thousands of donors.Advantageously, these transferrins result from the purification productobtained from blood of at least 1000 donors. Thus transferrin pools arereadily accessible. And indeed nowadays pooled transferrins consist ofhemoderivatives deemed as having no therapeutical or clinical uses. Theyare merely discarded.

Though no relationship has so far really been established between thegenetic diversity of transferrin and the major histocompatibilitycomplex (MHC) system in man, the serological detection and confirmationof the presence of a sufficient number of the dominant and relevant HLAcan nonetheless be relied upon to verify whether plasma pools from whichthe corresponding pooled transferrins are to be obtained originated froma sufficient number of donors. For instance a preferred starting plasmapool should prove to contain at least 4 serologically determinableantigens of each of the so-called HLA-A, HLA-B, HLA-C, HLA-D and HLA-DRseries. Reference is for instance made to FIG. 3.1, page 70 of the booktitled “Medical Immunology” edited in 1979 by James Irvine, TeviotScientific Publications, Ediburgh, Great Britain.

It has now been discovered that the immune tolerance inducing propertiesof transferrin are in fact concentrated in their glycan moieties.

Thus the present invention concerns more particularly a biologicalcomposition whose active principle consists of one or severaltransferrin-derived glycans, substantially free of transferrinpolypeptide moieties, said active principle comprising enough of thephenotypic information required to ensure an induction of tolerance in agiven immuno-depressed recipient host administered with said biologicalcomposition and grafted with antigens of a given donor host.

Preferred transferrin glycans are those which originate or areobtainable from the same mammalian species as the donor's.

The expression “transferrin glycans” as used throughout this patentdisclosure further extends to all glycans which display a substantiallysame profile as those directly obtained from transferrin, as evidencedby any of the analytical methods referred to in “Tools for Glycobiology”edited in 1994 by the Company known as Oxford GlycoSystems, availablefrom the Company itself in the U.S.A., i.e., Oxford GlycoSystems, Inc.,Cross Island Plaza, 133-33 Brookville Boulevard, Rosedale, N.Y. 11422,U.S.A., or from the European branch, i.e., Oxford GlycoSystems Ltd.,Hitching Court, Blacklands Way, Abingdon, OX14 1RG, UK.

Transferrin glycans as such have also been extensively studied:reference is made, of course in a non-limitative manner to generalpublications describing them, for instance the publication titled“Comparative study of the primary structures of sero-, lacto- andovotransferrin glycans from different species” of Geneviève Spik et al.,in Biochimie 70 (1988) 1459-1469, which describes transferrin glycansobtained from various mammalian species.

As glycoproteins, all transferrin of human and animal origin containcarbohydrates in amounts varying from 2 to 12%. Human serum transferrinhas been found to present a microheterogeneity based on the existence ofbi- and triantennary glycans of the N-acetyl-lactosaminic type. Threecarbohydrate molecular variants of transferrins could be distinguished:Tf-I (less than 1%) containing two triantennary glycans, Tf-II (approx.17%) with one triantennary and one biantennary glycan and Tf-III(approx. 82%) containing two biantennary glycans. The relativeproportions of these variants were found to change in women in the lasttrimester of pregnancy, the variants I and II showing an increase incontrast to variant III, which was found to decrease to approx. 67% (seeLeger et al. referred to hereafter). In addition, it has beenestablished, that human sero-transferrin contains two asparagineglycosylation sites in the C-terminal part of its single polypeptidechain and that the glycans are fully sialytated and not fucosylated.Like the corresponding transferrin, the glycans which can be obtainedtherefrom display similar microheterogeneities.

Detection of the mammalian species from which particular transferringlycans originate can be carried out by any person skilled in the art,e.g., by reaction of these glycans with sets of antibodies previouslyobtained against glycans of a number of different mammalian species,among which presumably that of the species from which the glycans understudy may originate. By way of non-limitative illustration, recourse canbe had to a method of the type disclosed in the publication titled“Physiological significance of the marked increased branching of theglycans of human serotransferrin during pregnancy” of Didier Léger etal. in Biochem. J., 257: 231-238 (1989) (Printed in Great Britain).

For instance transferrin glycans obtained from a human may be detectedby an immunological reaction with antibodies specific to humanglycans—or even to the corresponding human transferrins and then withhorse-radish-peroxidase—conjugated second antibodies raised againsttlg6, in accordance with Trowbridge et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 79: 1175-1179 and Burnette et al. (1981) Anal. Biochem. 112:195-203.

Of course other types of reactions can be envisaged for the samepurpose, e.g., by comparative analysis of the electrophoretic behaviorof the glycans under study and standards obtained from transferrinsthemselves obtained from different mammalian species.

A preferred composition according to the invention comprises the glycansobtained from transferrins also obtained from the donor of the antigensto be transplanted in the recipient host. These glycans can be obtainedfrom said transferrin by any of the well known methods applicable to theremoval of the polypeptide moieties and recovery of the correspondingglycans, e.g., a method of hydrazinolysis such as disclosed in thepublication of S. Takasaki et al. titled “Hydrazinolysis ofAsparagine-Linked Sugar Chains to Produce Free Oligosaccharides in“Methods of Enzymology (1982) Vol.83: 263-268, or in the publication ofT. Patel et al., titled “Use of Hydrazine to Release in Intact andUnreduced Form both N- and O-Linked Oligosaccharides from Glycoproteins”in Biochemistry (1993) 32:679-693; or by enzymatic cleavage in thepresence of a neuramididase or an endoglycosidase activity, such as thatproduced by Flavobacterium meningosepticum, as disclosed by J. H. Elderet al. (1982) in the publication titled “Endo-β-N-AcetylglucosaminidaseF: Endoglycosidase from Flavobacterium meningosepticum that cleaves bothhigh-mannose and complex glycoproteins” in Proc. Natl. Acad. Sci.U.S.A., Vol. 79:4540-4544, August 1982, or in the presence of theendo-β-N-acetylglucosaminidase F (Endo F) or peptide: N-glycosidase F(PNGase F) also obtainable from cultures of Flavobacteriummeningosepticum as disclosed by A. L. Tarentino et al. in thepublication titled “Deglycosylation of Asparagine-Linked Glycans byPeptide: N-Glycosidase F”.

Reference can also be made to the techniques generally disclosed in“Tools for glycobiology” supra.

However like in the case of transferrin, preferred biologically activecompositions include pooled transferrin-derived glycans obtained from anumber of donors sufficient to allow said pooled transferrin-derivedglycans to contain all the phenotypic information required to ensure foran induction of immune specific tolerance against antigens of adetermined allogeneic or xenogeneic donor in an immuno-depressed hostgrafted with said antigens, after that host had been administered anamount of such pooled transferrin-derived glycans effective to inducesaid immune specific tolerance.

Pooled transferrin-derived glycans of human origin can be obtained fromtransferrins which are themselves available in the trade: see “Tools forGlycobiology” already of record. Human transferrin-derived glycans,substantially free of the transferrin polypeptide moieties areavailable, e.g., at Oxford Glyco Systems, Inc.

BRIEF SUMMARY OF THE INVENTION

Like for the transferrin, the serological detection of a sufficientnumber of the dominant HLA antigens provides nonetheless an adequateverification system of whether the plasma pools from which thecorresponding pooled glycans are to be obtained originated from asufficient number of donors. For instance a preferred starting plasmapool should also prove to contain at least 4 serologically determinableantigens of each of the so-called HLA-A, HLA-B, HLA-C, HLA-D and HLA-DRseries.

It will further be appreciated that, as this will be further discussedhereafter, the results—e.g., those illustrated hereafter—obtainable withtransferrin-derived glycans also originating from the donor of theantigens are of great assistance in determining the degree of fitness ofpooled transferrin-derived glycans to achieve similar induction ofimmune tolerance against the antigens transplanted into a recipienthost. The closer the results produced in the same experimental protocolby the pooled glycans to the results obtained with glycans derived fromthe antigen donor himself, the better the “phenotypic matching” of thepooled glycans with the donor's organism.

It will also be readily apparent that the greater the number ofinstances in which a given composition containing pooled glycans willprovide as efficient an induction of immune tolerance in recipientsagainst antigens of different donors as the immune tolerances induced inthe same recipients by the corresponding “individual glycans” providedby the same donors respectively, the greater the “universality” of thepooled glycans of said given biological composition. This “universality”should be all the greater as the pooled glycans will also be originatingfrom a greater number of persons. It is then reflected by a similarcapability of the pooled transferrin glycans to induce in therecipients, in similar testing protocols, substantially the sameeffects, or effects of a same order of magnitude, as those achieved inthe recipients by the “individual glycans” also obtainable from therespective donors of the antigens whose transplantation into anyrecipient is to be achieved. The said effects e.g., are those describedmore fully in the examples which follow, i.e., the inability to“recognize” the donor lymphocytes and to mount an immediate- or adelayed-type immune response against the antigens of that donor, anabsence of cytotoxic antibodies in the “tolerant” recipient's serumagainst the donor's lymphocytes, an inability of “tolerant” recipient tomount a cell-mediated immune reaction in vivo against the donor'sleukocytes, an in vitro lack of reactivity of the splenocytes of the“tolerant” recipient towards irradiated donor's lymphocytes, etc.

It must further be appreciated that the expression “transferringlycans”, i.e., glycans essentially free of the transferrin peptidemoieties, do also cover but parts of these glycans, e.g., such glycansfreed of part or all of one of the antennary glycan chains when itincludes several of these chains, or partially desialylated chains, or asingle of these antennary glycan chains, in either sialylated, orpartially desialylated form, of course provided that the so-modifiedglycans or glycan parts do not loose their ability to induce theimmune-tolerance effects of the non-modified ones, e.g., as assayable bythe assay procedures disclosed in the examples.

The invention also concerns the combination of the pooledtransferrin-derived glycans and of at least one immunosuppressive drug,e.g., prednisolone, cyclophosphamide, cyclosporin, FK-506 ormethotrexate, particularly for use in a human host under the appropriatesequence of administrations, to induce immune tolerance in the hostagainst allogeneic or xenogeneic antigens to be grafted in said host.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an illustration depicting the use of transferrin-derivedglycans in conjunction with immunosuppression of the induction oftransplant tolerance.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

As will be seen hereafter the immuno-depression can be achieved by anadministration to the host of an immunosuppressive drug, e.g.,cyclosporin, prednisolone, cyclosphosphamide, etc. or by irradiation.

It has been found that in animals complete destruction of theimmune-system of the recipient host undergoing bone marrowtransplantation may not be necessary. The use of chemicalimmunosuppressants, in conjunction with the pooled transferrin-derivedglycans, in the appropriate sequences of administration as discussedhereafter may thus be preferable to lethal irradiation. In leukemicpatients already undergoing an immunosuppressive chemotherapy,allogeneic or xenogeneic bone marrow grafting, where hold appropriate,may even no longer require an additional administration of animmunosuppressant, in conjunction with both the administration of thepooled transferrin-derived glycans and the transplantation of bonemarrow cells. Partial or total body irradiation as preferably proposedin the grafting protocols proposed by the Applicant in his earlierpublications or patents may no longer be required.

After administration of the appropriate pooled transferrin glycanstransplantation of bone marrow may be replaced by transplantation ofallogeneic or xenogeneic peripheral blood progenitor cells mobilizedfrom the donor's bone marrow into the peripheral blood by administrationof cytokines like granulocyte-macrophage colony stimulating factor(GM-CSF) or Multipotential-CSF (Interleukin-3) and harvested byleukapheresis using a cell separator system.

In the case of organ transplantation, donor's antigens to be initiallypresented to the recipient host in combination with the appropriatetransferrin glycans before transplantation of the organ itself mayconsist of the “buffy coat” or leukocytes from the donor's peripheralblood after centrifugation, containing granulocytes and lymphocytes withHLA-specific antigenic markers of the individual donor. As with bonemarrow transplantation, the donor's buffy coat may also be replaced byallo- or xenogeneic peripheral blood progenitor cells, mobilized intothe peripheral blood and harvested as described. Most preferably, thispresentation of the antigens is done after chemical immunosuppression atthe bottom line of suppression of the immune system and before theendogenous reconstitution starts. The stage of immunosuppression can beevaluated by leukocyte counts in the peripheral blood. Apparently bestresults are obtained when the administration of transferrin glycans andthe initial presentation of donor antigens take place just at thebeginning of the endogenous reconstitution of the immune system, whichfollows the chemical immuno-suppression. The early presentation oftransferrin glycans (donor-type or pool) together with antigens (e.g.,at day 3 in the mouse model of FIG. 1 reported hereafter) will producetolerance in said human-to-mouse model. Therefore important elements ofthis invention comprise the administration of transferrin glycans(donor-type or pool) and the timely injection of specific donorantigens, which will induce possibly a selection or deletion of immunereactive cells and thus a specific immune tolerance, both in theallogeneic as well as in the xenogeneic approach.

The same considerations do of course apply to other kinds of antigens.Needless to say that the timing and sequence of administration will haveto be studied in each case. The organ to be grafted should not beadministered too late after the immunosuppression and the initialpresentation of transferrin glycans and antigen, particularly whenantigen reactive cells will already have been produced again by therecipient host organism. In such event, the recipient host organism mayno longer become tolerant.

The invention is not limited to human pooled transferrin-derivedglycans, particularly for the above-mentioned uses. It also extends topooled transferrin-derived glycans of animal origin, particularly foruse in conjunction with the grafting even in man of xenogeneic cells,tissues or organs obtained from the same animal species as the pooledtransferrin.

[A] Preparation of Transferrin (Tf)

A pool of human plasma (ca. 1000 donors), iron saturated with Fe³⁺according to Bates G. W. et al, J. Biol. Chem. 1973, 248:3228-32, isdiluted in phosphate buffer and diafiltered on hollow fibers, cut-off30,000 to remove Fe³⁺ excess, stored one night at 4° C. and filteredthrough 0.45 μm sterile membranes. The purification procedure consistsof two chromatographic steps on ion exchangers, by using buffers atsuitable ion strength and pH, in order to selectively removecontaminants such as albumin and immunoglobulins and hence to elute Tfwith a purity >95%. After diafiltration to re-establish physiologicalsalt conditions, the solution of apo- or iron-saturated Tf isfreeze-dried.

Extraction procedures of transferrins are well known. Some of them arerecalled in Applicant's earlier patent EP 0426924 or in the ClinicaChimica Acta publication already referred to hereabove.

Human pooled transferrins may for instance be obtained as disclosedhereinafter.

[B] Preparation of Transferrin-Derived Glycans

Glycans are isolated from human Tf-pool (ca 1000 donors), by thehydrazinolysis method carried out as disclosed in the publication of S.Takasaki et al. referred to above. 1 mg Tf pool contains approximately20 μg of glycans. In all experiments reported hereafter glycans wereused at a dose of 5 μg/mouse i.p., which corresponds to glycans contentin the usual dose of Tf (200 μg/mouse) in previous experiments.

[C] Induction of Transplantation Tolerance

The experimental protocols which have been used are briefly recalledhereafter, prior to being set forth subsequently in a more detailedmanner.

Prednisolone (Pr) and cyclosphosphamide (Cy) were chosen asimmunosuppressants; their respective dosages were adjusted in differentmouse strains according to changes in their immunological parameters. Byusing this model, the first indications of the tolerance-inducingactivity of Tf-glycans were observed in preliminary studies on theimmune response of mice to human erythrocytes. It was found that humanTf-glycan treatment in immunosuppressed and antigen treated miceinhibits the primary and the secondary immune response to human redblood cells (HRBC)(Tables 2A and 2B).

Since histocompatibility antigens are presented predominantly onleukocytes, it was important to know whether Tf-glycan treatment caninduce donor-specific transplantation tolerance in mice injected withperipheral blood “buffy coat” leukocytes. In fact, the abrogation of acell-mediated immune response towards the Tf-glycan donor tissueantigens was demonstrated with the popliteal lymph node assay inchemically immunosupressed mice treated with Tf-glycan of the donor.

Also absence of donor-specific antibody- and complement-mediatedcytotoxicity of mouse serum towards human lymphocytes in chemicallyimmunosuppressed mice treated with human individual Tf-glycans or pooledTf-glycans was confirmed by trypan-blue exclusion assay (Tables 6A and6B).

From the data presented here, it is possible to see that also theadministration of glycans from a human plasma pool, combined withcritically timed presentation of individual specific cell antigens(leucocytes) can produce a state of immunological tolerance. Besidestolerance-inducing properties, human pooled Tf-glycans also possess aremarkable immunoprotective activity by preventing thymus involution andlymphopenia and by increasing the survival rate of chemicallyimmunosuppressed mice.

The above mentioned human-to-mouse model and the results point to aclear-cut tolerance-inducing effect of human glycans by sequentialand/or combined administrations of pooled Tf-glycans and cell antigens.Individual Tf-glycans alone are not immunosuppressive per se and areunable to produce tolerance to human antigens in the mouse.

The implication deriving from the disclosed models are obvious. Suchtransplantation system should be adaptable to larger mammals and to man.In addition, the understanding of the mechanism by whichtransferrin-derived glycans from plasma pools produce tolerancecertainly deserves intensive investigations for a possible adaptation ofthe model to a number of pathological conditions such as cancer,autoimmune disease, immunodeficiency diseases (e.g., AIDS), geneticdefects or diabetes.

In order to fully illustrate the present invention and advantagesthereof, the following specific examples are given, it being understoodthat the same are intended only as illustrative and in nowiselimitative.

In the course of the description the following abbreviations were used.

ABBREVIATIONS

Ag=antigen

BM=bone marrow

BMT=bone marrow transplantation

Con A=concanavalin A

Cy=cyclophosphamide

HRBC=human red blood cells

HSA=human serum albumin

IS=immunosuppression

i.p.=intraperitoneally

MTT=(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrasolium bromide)

OD=optical density

PLNT=popliteal lymph node test

Pr=prednisolone acetate

Tf=transferrin

Tf glycans=glycans obtained from transferrins

Materials and Methods

Animals

Adult inbred, 3 to 8 month-old, male or female C57BL/6 or BalB/c micemaintained in our animal quarters under conventional conditions wereused. The mice received water and fodder ad libitum and the roomtemperature was 21-22° C.

Immunosuppressive Drugs for Immunosuppression (IS)

Prednisolone acetate (Pr) was purchased from FLUKA Inc., Buchs,Switzerland. It was injected once i.p. as a 1:10 ethanol-watersuspension immediately after preparation. Cyclophosphamide (Cy) waspurchased from FLUKA and dissolved with water shortly before i.p.injection. Doses and schedule of injection were as indicated in thesingle experiments.

Preparation and Purification of Transferrin (Tf)

Ferric sulphate and nitrilotriacetic acid (purity 99.5%) used forferric-nitrilotriacetate preparation was from SIGMA. Sodium bicarbonatewas from Merck (Darmstadt, Germany). Salts used for buffers were fromCarlo Erba (Milano, Italy, Pharmaceutical type). Diafiltration andconcentrations were carried out in a tangential flow system, withultrafiltration membranes cut-off=30,000. DEAE-Sepharose andCM-Sepharose were from Pharmacia (Uppsala, Sweden). The chromatographicsystem was based on preparative chromatographic columns connected with aperistaltic pump P1, monitor UV and recorder from Pharmacia. Virusinactivation was performed in a thermostatic-controlled water bath.Total proteins were measured by the method of Lowry et al. against astandard of bovine serum albumin (Pierce, Amsterdam, Netherlands).Antigen protein was determined by radial immunodiffusion withNor-Partigen Tf and serum protein standard from Behring (Marburg,Germany). Agarose gel electrophoresis was performed with a Helenamillipore (Milford, USA) 625 LC chromatograph; the conditions were thefollowings: column TSK3000SW (75 mm×600 mm); flow rate 0.8 ml/min;mobile phase phosphate 0.05 M, sodium chloride 0.15 M, Na N3 0.05%, pH7; detector UV Merck-Hitachi L4200 (wavelength 280 nm);integrator-recorder Perkin Elmer LCI-100 (Norwalk, USA). Thepurification process was as follows: plasma was initially saturated withiron by the addition of sodium bicarbonate and ferric-nutrilotriacetate(FENTA) according to Bates & Schlabach. Plasma was then diluted anddiafiltered against phosphate buffer 10 mM prior to loading onto acolumn with DEAE-Sepharose. The column was eluted with phosphate buffer50 mM, obtaining a Tf fraction contaminated by immunoglobulins. Thisfraction was diafiltered against phosphate buffer with a different pH,and loaded onto a column with CM-Sepharose. The flow-through was a Tffraction with a ≧95% purity. The Tf solution was concentrated andvirus-inactivated with a pasteurization step at 60° C. for 10 hours, inthe presence of suitable stabilizers. After diafiltration to eliminatethe stabilizers added for the heating treatment, the Tf concentrate wassterile-filtered, dispensed into vials, freeze-dryed and stored at +4°C. Tf obtained with this purification procedure was apo-Tf. To obtainiron-Tf, an additional saturation step with ferric-nitrilotriacetate anddiafiltration for eliminating free ferric ions were used. Acceptableendotoxin values in Tf preparations were below 0.7 ng/mg (Limulusassay).

a) Drug-induced Immunosuppression and Evaluation of its Efficacy andDuration

The basic idea underlying our model was that the presentation of Tf orTf-glycans from a species- or strain-different donor must take place ina condition of complete or very deep depletion of mature immunocytes andbefore or at the beginning of endogenous reconstitution or repopulationof immune cell producing organs (e.g., the thymus or the bone marrow),combined with presentation of donor- and Tf-matched or Tf-glycan-matchedantigens in the course of regeneration and maturation of immunocytes inthe BM and in the thymolymphatic tissues (see FIG. 1: the “empty mouse”model). We thought that administration of Tf or Tf-glycans must continueat the time of, and after immunization, until all putativeantigen-reactive cells have maturated to a stage leading to specifictolerance or to areactivity-unresponsiveness. For this purpose numerousexperiments were initially carried out with mice in order to establishthe most suitable type of immunosuppressive treatment and schedule ofinjection which would induce a durable and profound cytolytic andcytotoxic effect on thymic and BM cell populations and consequently atemporary abrogation of antibody production and cell-mediated immunereactions. Several drugs alone or in combination were studied for theircapacity to produce a temporary but deep depression of immunity as e.g.,busulphan, azathioprine, cyclosporin, methotrexate, cyclophosphamide,prednisolone. At the end of our long-term trials, a combination ofprednisolone acetate (Pr) and cyclophosphamide (Cy) was chosen whichproduced a profound and durable immunosuppression (IS) for two-threeweeks without severe side-effects and elevated mortality. As can be seenin Table 1, the IS with a combination of Pr and Cy resulted in an almostcomplete BM cell depletion at day 3 and day 4 of the experiment. Thisdepletion was more prolonged when the mice received 200 mg/Kg Cy. Inthis case the reconstitution started at day 5 (Table 1). It is alsovisible that IS produced a drastic reduction of peripheral bloodleukocytes which were most reduced at day 4 and day 5 of the experiment.

TABLE 1 DEPLETION AND RECOVERY OF NUCLEATED CELL NUMBER IN THE BONEMARROW OR IN PERIPHERAL BLOOD OF MICE AFTER CHEMICAL IMMUNOSUPPRESSION(IS) Cell counts Leukocytes (× 10⁶/ml) Lymphocytes (× 10⁶/ml) Tibia^(b)(× 10⁶) after IS^(a) 100 mg/kg Cy 200 mg/kg Cy 100 mg/kg Cy 200 mg/kg Cy100 mg/kg Cy 200 mg/kg Cy day 0^(c) 5.20 ± 0.70 5.20 ± 0.70 3.60 ± 0.503.60 ± 0.50 7.00 ± 1.40 7.00 ± 1.40 day 2 2.40 ± 0.40 0.60 ± 0.20 1.10 ±0.20 0.15 ± 0.06 1.20 ± 0.20 1.00 ± 0.03 day 3 1.70 ± 0.40 0.50 ± 0.100.70 ± 0.14 0.14 ± 0.02 1.00 ± 0.03 0.50 ± 0.03 day 4 0.90 ± 0.50 0.20 ±0.10 0.30 ± 0.15 0.07 ± 0.03 1.10 ± 0.10 0.50 ± 0.10 day 5 0.70 ± 0.500.02 ± 0.02 0.46 ± 0.30 0.01 ± 0.00 2.00 ± 0.30 1.20 ± 0.02 day 6 1.60 ±0.00 0.40 ± 0.20 1.00 ± 0.35 0.20 ± 0.10 3.40 ± 0.40 1.50 ± 0.10 day 75.50 ± 0.70 1.20 ± 0.60 2.80 ± 0.20 0.60 ± 0.20 5.50 ± 0.90 3.40 ± 1.00^(a)IS — Pr at the dose of 90 mg/Kg and Cy at the dose of 100 mg/Kg or200 mg/Kg were injected on day 0. The injection of Cy was repeated atthe same dosage on day 1. ^(b)The BM cells were suspended in 0.5 mlmedium from single tibiae and counted. ^(c)-before immunosupression. Thevalues are Means ± SE. For each point three mice were used.

b) Effect of Tf-glycans on Antibody Production to Human Cell Antigens inIS Mice

Many experiments and trials in the course of four years preceded theconclusive model illustrated in FIG. 1. In order to develop thepresently adopted model for induction of xeno-“tolerance”(human-to-mouse), human Tf-glycans were used.

The following assays, which aim at evaluating the effect of Tf-glycanson the secondary immune response of mice to human red blood cells (alsoobtained from the donor whose “buffy coat” leukocytes were used forimmunization ) were performed in chemically immunosuppressed mice.

Balb/c mice were chemically immunosuppressed by injecting 90 mg/kgprednisolne (Pr) and 100 mg/kg cyclophosphamide (Cy) i.p. on day 0. Onday 1, 100 mg/kg Cy were injected again i.p. The immunosuppressed micewere daily injected i.p. from day 3 to 9 and from day 16 to 18 with 200μg Tf pool purified from human blood plasma pool in 0.5 ml (Group 2) orwith 5 μg Tf-glycans purified from human Tf pool (Group 3) or received0.5 ml saline i.p. (Group 4). All the mice including non-IS Group 1 wereimmunized twice i.p. with human antigens, namely 1.0×10⁶ humanperipheral blood “buffy coat” leukocytes in 0.3 ml on day 3 and on day16. The bleeding was performed on day 31 of the experiment, serumsamples were collected and stored at −70° C. until the assay.

The results are reported in Tables 2A and 2B hereafter.

TABLES 2A and 2B

TABLE 2A INDUCTION OF DONOR-SPECIFIC TOLERANCE WITH GLYCANS EFFECT OFGLYCANS ON THE SECONDARY IMMUNE RESPONSE TO HUMAN RED BLOOD CELLS(HRBC)° IN CHEMICALLY IMMUNOSUPPRESSED MICE. (Exp. 36, day 31) −log₂antibody titers Groups secondary response 1. normal 8.0 ± 0.3 immunizedcontrol n = 5 2. IS + Tfpool_(human) 6.0 ± 1.5 n = 3 3. IS + Glycans 4.0 ± 0.6* n = 4 4. IS + saline 8.3 ± 0.3 n = 3 IS—immunosuppresion;Tf—transferrin; n—number of mice per group; *p < 0.05 vs Gr. 1,4

TABLE 2B INDUCTION OF DONOR-SPECIFIC TOLERANCE WITH GLYCANS EFFECT OFGLYCANS ON THE SECONDARY IMMUNE RESPONSE TO HUMAN RED BLOOD CELLS(HRBC)° IN CHEMICALLY IMMUNOSUPPRESSED MICE. (Exp. 37, day 30) −log₂antibody titers Groups secondary response 1. normal 8.0 ± 0   immunizedcontrol n = 4 2. IS + Tfpool_(human) 5.8 ± 0.6 n = 5 3. IS + Glycans 3.8 ± 0.6* n = 5 4. IS + saline 6.4 ± 0.5 n = 8 IS—immunosuppresion;Tf—transferrin; n—number of mice per group; *p < 0.01 vs Gr. 1,4

The results obtained indicated that injection of Tf-glycans from humanplasma pool into IS mice resulted in a considerable lastingunresponsiveness or marked decrease of production in most of the mice ofantibodies against randomly chosen donors of human peripheral blood cellantigens. It can be seen (Tables 2A and 2B) that injection of viablehuman peripheral blood cells into the IS and human Tf-glycans treatedmice resulted in lasting inhibition of the immune response to humanantigens. No abrogation or lasting diminution of the antibody responsecould be seen when the IS and human-antigen-immunized mice had beeninjected with saline in the course of the immune restoration after IS.

In summary, these preliminary findings served only to establish thatpresentation of both human Tf or human transferrin-glycans from plasmapool and individual-specific human cell antigens in the IS murine hostresulted in a durable inability of most of the mice to mount a normalimmune response to human antigens;

c) Human Tf-glycans Alone do not Exert an Immunosuppressive Effect inMice

Repeated injections of human glycans into mice did not impair ordecrease their primary or secondary (memory) response to humanerythrocytes and no effect could be observed on peripheral bloodleukocyte count and on the delayed type hypersensitivity response(Oxazolone test). Human Tf glycans do not exert themselves animmunosuppressive activity in mice (data not reported here).

The absence of direct immunosuppressive effects induced by Tf-glycans aswell as their inability of affecting both the antibody response and thecell-mediated immunity in normal mice are reflected by the resultspresented in Tables 3 and 4 hereafter, as a result of assays which werecarried out as follows.

10% human erythrocytes in 0.2 ml i.p. on day 3 and on day 9.

Balb/c mice (3 animals/group) were daily injected intraperitoneally(i.p.) for 10 days with 5 μg of glycans in 0.5 ml (Group 1). Group 2 wassimilarly injected with saline while Group 3 was left untreated. On day3 and again on day 9, all mice were immunized with 10% human erythrocytesuspension in 0.2 ml.

The bleeding was performed on day 9 and on day 14 of the experiment. Theprimary and the secondary immune response were measured by using thedirect hemagglutination assay.

As apparent from the results displayed in Table 4, glycans fromtransferrin have no immunosuppressive effects and do not affect theantibody response in mice.

The effect of glycans on the primary and the secondary immune response(IR) to human red blood cells (HRBC) in normal mice was tested uponusing the following protocole.

HRBC—10% human erythrocytes in 0.2 ml i.p. on day 3 and on day 9. Balb/cmice (3 animals/group) were daily injected intraperitoneally (i.p.) for10 days with 5 μg of glycans in 0.5 ml (Group 1). Group 2 was similarlyinjected with saline while Group 3 was left untreated. On day 3 andagain on day 9, all mice were immunized with 10% human erythrocytesuspension in 0.2 ml.

TABLE 3 Effects of glycans or the primary and secondary immune response(IR) to human red blood cells (HRBC) in normal mice. N mouse −log₂ Abtiters Groups serum primary IR secondary IR Gr. 1 1. 6.0 6.0 HRBC +glycans 2. 6.0 8.5 n = 3 3. 6.0 8.0 Mean ± SE 6.0 ± 0   7.5 ± 0.8 Gr.2 1. 6.0 8.0 HRBC + saline 2. 4.0 7.0 n = 3 3. 6.0 9.0 Mean ± SE 5.3 ±0.6 8.0 ± 0.6 Gr. 3 1. 6.0 8.0 HRBC 2. 5.0 7.0 n = 3 3. 6.0 7.0 Mean ±SE 5.7 ± 0.3 7.3 ± 0.4

Similar results appear in Table 4, which show that glycans obtained fromsingle transferrin batches and glycans from human transferrin poolneither have immunosuppressive effects nor affect cell-mediated immunityin normal mice.

TABLE 4 Effect of Glycans on the primary and secondary delayed-typehypersensitivity (DTH) response in normal mice No Ear thickness (× 0.01mm) Group of mice before Ch-1 after Ch-1 Δ_(I) before Ch-2 after Ch-2Δ_(II) Gr. 1 1. 25.0 27.5 2.5 24.5 28.5 4.0 Glycans 2. 24.5 27.5 3.024.0 26.0 2.0 3. 25.5 28.5 3.0 25.5 29.0 3.5 Mean ± SE 2.8 ± 0.2 3.2 ±0.6 Gr. 2 1. 24.5 27.5 3.0 25.5 27.5 2.0 saline 2. 25.5 28.5 3.0 26.028.5 2.5 3. 25.0 29.0 4.0 26.5 28.5 2.0 Mean ± SE 3.3 ± 0.3 2.2 ± 0.2Gr. 3 1. 24.5 28.0 3.5 25.0 29.0 4.0 untreated 2. 25.5 28.5 3.0 26.028.0 2.0 3. 24.5 27.5 3.0 25.0 28.0 3.0 Mean ± SE 3.2 ± 0.2 3.0 ± 0.6Ch-1 — Challenge 1 (on day 7); Ch-2 — Challenge 2 (on day 14)

Additional assays, i.e., the mixed lymphocytes culture (MTT assay) andthe Trypan blue exclusion assay for measurement of complement-mediatedcytolysis, as well as the results obtained are reported hereafter. Thegeneral procedures are reported first.

Mixed Lymphocyte Culture (MTT Assay)

a) Culture Medium

Spleen cells from the experimental mice were cultured in a medium withthe following composition: 50% RPMI 1640 (SIGMA); 30% Dulbecco'smodification of Eagle's medium (DMEM, Seromed, Berlin, Germany); 10%Iscove's modification of Dulbecco's medium (IMDM, Seromed); 10% fetalcalf serum (FCS, Seromed); 2 mM glutamine, 100 units/ml Penicillin, 100μg/ml Streptomycin (SIGMA); 29 μM 2-mercaptoethanol (FLUKA).

b) Isolation of Mouse Spenocytes

The mice were sacrificed by cervical disclocation, the spleens removedin aseptical conditions and immersed into RPMI 1640 medium. Each spleenfrom the experimental mice was teased separately in a Potter glass withTeflon pestle and the splenocyte suspensions from individual mice wereprepared in the culture medium. Erythrocytes were eliminated by osmoticshock: the spienocyte suspension was added to the same volume ofbidistilled water. After 5 min the mixture was diluted with the culturemedium containing double NaCI concentration. After centrifugation (1500rpm, 10 min, room temperature) the supernatant was removed and the cellswere resuspended with the culture medium to the final concentration of2×10⁶ cells/ml. This suspension was distributed into 96-well plates withflat bottom (Falcon, Oxnard, USA) at 100 μl (2×10⁵ cells) per well.

c) Isolation of Human Lymphocytes Target, Target Cell Irradition CellIrradiation and Preparation of Samples

Human lymphocytes from heparinized blood of individual donors wereisolated by density gradient centrifugation (2000 rpm for 20 min at roomtemperature) by using Accuspin System Histopaque 1077 (SIGMA). Aftercentrifugation the supernatant was removed and the cells were washedthree times with the culture medium and resuspended in this medium tothe final concentration of 2×10⁶ cells/ml. The human lymphocytes as wellas a part of the mouse splenocytes were irradiated with a total dose of2500 Rad by using a Gammacell 3000 Elan installation (Nordion Intern.Inc., Canada). The irradiated cells were added to the mouse splenocytesin a 100 μl volume (2×10⁵ cells) per well. The final culture wellcontained 2×10⁵ mouse splenocytes and 2×10⁵ irridiated cells (humanlymphocytes or mouse splenocytes) in a 200 μl volume.

The following cell combinations were used for the mixed lymphocytesculture: A—splenocytes from experimental mice (responding cells);A*—irradiated mouse splenocytes from intact mice of the same strain, sexand age; B*—irradiated human lymphocytes (stimulating cells). Thefollowing combinations were included: 1. A+B* (stimulation of mousesplenocytes by human antigens); 2. A+A* (background or non-specificactivity); 3. A+ConA (ability of mouse spienocytes to respond). In thelast case 10 μl (10μl/ml) ConA (SIGMA) were added to each wellcontaining A cells, so that the final volume of this combination was 110μl per well. A+B and A+A* combinations were evaluated in 4-5 parallelwells for each individual cell mixture suspension A+ConA combination wasevaluated in two parallel wells for each splenocyte suspension.

The cell combinations were maintained in a humidified atmosphere of 5%CO₂—95% air at 37° C. for 5 days. On day 2 of culture, 100 μl of mediumwere eliminated from all wells except the (A+ConA) combination, and 100μl of fresh culture medium were added to all wells.

The MTT Assay

The assay is dependent on the cellular reduction of MTT by themitochondrial dehydrogenase of viable cells to a blue formazan productwhich can be measured spectrophotometrically.

The MTT assay was performed according to Mossman with somemodifications. MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrasoliumbromide) (SIGMA) was dissolved in phenol red-free RPMI 1640 (SIGMA) at afinal concentration of 0.5 mg/ml (MTT stock solution) filtered through a0.22 μm filter and used on the same day. After 5 days of culture themedium was removed from the plates by aspiration and 100 μl aliquot ofMTT stock solution was added to each well of the assay plates which werethen incubated for further 4 hours at 37° C. in the CO₂ incubator. Thenthe supernatant from the wells was removed by aspiration.Acid-isopropanol (100 μl of 0.04 N Hcl in isopropanol) was added to allwells and mixed thoroughly to dissolve the dark blue crystals. Ten tofifteen min later the plates were read with a Microelisa Reader (SRATautomatic, 90139, Austria) using a test wavelength of 570 nm andreference wavelength of 690 nm.

Optical density (OD) of the A+B* combination corresponds to intensity oftwo components: background activity of mouse splenocytes and theirproliferative reaction to human antigens, while OD of the A+A*combination measures the background activity of mouse splenocytes understudy. The difference between OD_(A+B) and OD_(A+A*) was considered asthe true reaction of mouse splenocytes to human antigens. To combine theresults of similar experiments, the magnitude of this reaction wasexpressed as percent. The mean value of the reaction innon-immunosuppressed and immunized mice was established at 100%.

Trypan Blue Exclusion Assay for Measurement of Complement-mediatedCytolysis

Lymphocytes were isolated from heparinized blood of human donor by theuse of Accuspin System Histopaque 1077 (SIGMA, St. Louis, USA). Thecells were washed tree times with isotonic saline and resuspended inconcentration 4-6×106 cells/ml. Samples of mouse serum (heat inactivatedat 56° C. for 30 min) were dispensed in 25 μl volume. An equal volume ofthe human lymphocyte suspension was added to each tube and the mixturewas incubated at 37° C. for 30 min. Then 50 μl rabbit complement (RabbitHLA-ABC, SIGMA) was added to each sample, and incubation was carried outfor 10 min at 37° C. Controls were set up using normal mouse serum orsaline to ensure that the complement is non-toxic. After centrifugation(2000 rpm for 5 min) the supernatant fluid was removed from each tubeleaving the cell pellet at the bottom and tubes were immersed into ice.Before reading, 25 μl Trypan Blue solution (SIGMA) was added to eachtube and the proportion of stained (non-viable) cells was counted.

Mixed Lymphocyte Culture (MTT Assay)

The data reported in connection with this assay demonstrate that thecombined administration of human Tf-glycan-pool and, as antigen,individual human leukocytes in the IS mice induce a profound or completeareactivity of the effector mouse spleen lymphocytes towards the targetlymphocytes from the human donor. In fact the activation values in vitroare comparable to those of normal, non-IS and non-immunized mice.

The conditions under which the assays were carried out (legend whichfollows) and the results obtained appear in Table 5A.

Legend to Table 5A Mixed Lymohocytes Culture (MTT assay) on Day 65 ofthe Experiment

Balb/c mice were chemically immunosuppressed by injecting 90 mg/kgprednisolone (Pr) and 100 mg/kg cyclosphosphamide (Cy) i.p. on day 0. Onday 1, 100 mg/Kg Gy were injected again i.p. The immunosuppressed micewere daily injected i.p. from day 3 to day 9 and from day 16 to day 18with 200 μg Tf pool purified from human Tf pool in 0.5 ml (Group 1) orwith 5 μg glycans purified from human Tf pool (Group 2) or received 0.5ml saline i.p. (Group 3). All the mice including non-IS Group 4 wereimmunized twice i.p. with human antigens, namely 1.0×106 humanperipheral blood “buffy coat” leukocytes in 0.3 ml on day 3 and on day16. The mice were killed on day 65 of the experiment and mixedlymphocyte culture reaction of mouse splenocytes against humanlymphocytes (from the same donor whose “buffy coat” leukocytes were usedfor immunization) was performed and evaluated by using MTT assay (seeMixed lymphocytes culture, MTT assay).

TABLE 5A INDUCTION OF TRANSPLANTATION TOLERANCE WITH Tf POOL AND GLYCANSMIXED LYMPHOCYTE CULTURE REACTION (MTT-TEST) DAY 65 OF THE EXP. 37OD_(A+B*) OD_(A+A*) Δ Groups I II (I − II) 1. IS + AgV.L. + Tf pool0.213 ± 0.040 0.165 ± 0.026 0.048 ± 0.021° (n = 3) 2. IS + AgV.L. +glycans 0.214 ± 0.017 0.142 ± 0.009 0.073 ± 0.009° (n = 3) 3. IS +AgV.L. + saline 0.244 ± 0.036 0.153 ± 0.040 0.092 ± 0.010 (n = 3) 4.non-IS + AgV.L. 0.243 ± 0.063 0.110 ± 0.047 0.133 ± 0.044 (n = 5) intact(n = 1) 0.140 0.082 0.058 A — splenocytes from individual experimentalmice (2 × 10⁵ cells/well), five wells for each mouse in parallel. B* —irradiated (2500 rad) lymphocytes of V.L. (2 × 10⁵ cells/well) A* —irradiated (2500 rad) syngeneic splenocytes from intact Balb/C mice (2 ×10⁵ cells/well) OD — optical density with a 570 nm test wavelength and a690 nm reference wavelength. Δ — the difference betweenOD_(experimental) and OD_(base line) Cell mixture was cultivated during5 days in CO₂ (5%) incubator °p_(U) < 0.05 vs Gr. 3 (non-parametricstatictics U-criterium Wilcoxon-Mann-Whitney)

Results of a same nature were obtained on day 103 of the experiment. Theconditions under which the assay was carried out are reiteratedhereafter

Mixed Lymphocyte Culture (MTT Assay) on Day 103 of the Experiment

Balb/c mice were chemically immunosuppressed by injecting 90 mg/kgprednisolone (Pr) and 100 mg/kg cyclophosphamide (Cy) i.p. on day 0. Onday 1, 100 mg/kg Cy were injected again i.p. The immunosuppressed micewere daily injected i.p. from day 3 to 9 and from day 16 to 18 with 200pg Tf pool purified from human blood plasma pool in 0.5 ml (Group 1) orwith 5 μg glycans purified from human Tf pool (Group 2) or with 200 μgHSA (Group 3) or received 0.5 ml saline i.p. (Group 4). All the miceincluding non-IS Group 5 were immunized twice i.p. with human antigens,namely 1.0×10⁶ human peripheral blood “buffy coat” leukocytes in 0.3 mlon day 3 and on day 16. The mice were killed on day 103 of theexperiment and the mixed lymphocyte culture reaction of mousesplenocytes against human lymphocytes¹ was performed and evaluated byusing the MTT assay.

¹lymphocytes were from the same donor whose “buffy coat” leukocyes wereused for immunization.

The results appear in Table 5B hereafter.

TABLE 5B INDUCTION OF TRANSPLANTATION TOLERANCE WITH GLYCANS MIXEDLYMPHOCYTE CULTURE REACTION (MTT-TEST) DAY 103 OF THE EXP. 36 OD_(A+B*)OD_(A+A*) Δ Groups I II (I − II) 1. IS + AgW.P. + Tf pool 0.157 ± 0.0100.130 ± 0.020  0.027 ± 0.012* (n = 4) 2. IS + AgW.P. + glycans 0.115 ±0.005 0.093 ± 0.017  0.022 ± 0.013* (n = 3) 3. IS + AgW.P. + HSA 0.140 ±0.031 0.062 ± 0.021 0.078 ± 0.010 (n = 2) 4. IS + AgW.P. + saline 0.172± 0.027 0.122 ± 0.058 0.050 ± 0.030 (n = 2) 5. non-IS + AgW.P. 0.175 ±0.016 0.085 ± 0.015 0.089 ± 0.012 (n = 4) intact (n = 1) 0.056 0.0120.044 A — splenocytes from individual experimental mice (2 × 10⁵cells/well), five wells for each mouse in parallel. B* — irradiated(2500 rad) lymphocytes of W.P. (2 × 10⁵ cells/well) A* — irradiated(2500 rad) syngeneic splenocytes from intact Balb/C mice (2 × 10⁵cells/well) OD — optical density with a 570 nm test wavelength and a 690nm reference wavelength. Δ — the difference between OD_(experimental)and OD_(base line) Cell mixture was cultivated during 5 days in CO₂ (5%)incubator *p < 0.05 vs Gr. 3; p < 0.02 vs Gr 5

Trypan-blue Exclusion Assay

The assay permits testing the permeability of cells after theirincubation with antibodies and complement. If cytotoxic antibodies bindto the membranes of target cells, complement is fixed and cellpermeability increases. It is used to assess cell permeability or“death” by adding a solution of trypan blue which penetrates into deadcells, but leaves viable cells unstained.

The assays were carried out upon using antigens of different persons (WPand VL).

The assay was made according to the following protocol:

Complement Dependent Cytotoxicity of Mouse Serum (Trypan Blue ExclusionAssay on Day 31 and on Day 61 of the Experiment)

Balb/c mice were chemically immunosuppressed by injecting 90 mg/kgprednisolone (Pr) and 100 mg/kg cyclophosphamide (Cy) i.p. on day 0. Onday 1, 100 mg/kg Cy were injected again i.p. The immunosuppressed micewere daily injected i.p. from day 3 to 9 and from day 16 to 18 with 200μg Tf pool purified from human blood plasma pool in 0.5 ml (Group 1) orwith 5 μg Glycans purified from human Tf pool (Group 2) or received 0.5ml saline i.p. (Group 3). All the mice including non-IS Group 4 wereimmunized twice i.p. with human antigens (Ag. WP and Ag. VL) namely1.0×10⁶ human peripheral blood “buffy coat” leukocytes in 0.3 ml on day3 and on day 16. The bleeding was performed on day 31 and on day 61 ofthe experiment, serum samples were collected and stored at −70° C. untilthe assay.

Two series of experiments were ran, the results of which appear inTables 6A and 6B hereafter.

TABLE 6A SPECIFIC ABROGATION OF THE IMMUNE RESPONSE WITH GLYCANSCOMPLEMENT DEPENDENT CYTOTOXICITY OF MOUSE SERUM EXP. 36 TRYPAN BLUEEXCLUSION ASSAY day 31 day 61 Lymph. Lymph. Lymph. Lymph. W.P. M.L. W.P.M.L. Groups % of dead cells 1. IS + AgW.P. + Tf pool 11.6 ± 4.7°  7.2 ±4.6* ND 7.8 ± 2.1° n = 7 n = 4 n = 6 2. IS + AgW.P. + glycans 7.6 ± 0.8°4.8 ± 0.9*° ND 6.7 ± 1.4° n = 9 n = 10 n = 8 3. IS + AgW.P. + saline43.4 ± 18.8 41.6 ± 14.4 ND 27.4 ± 14.0 n = 5 n = 7 n = 5 4. non-IS +AgW.P. 99.0 ± 0.3  97.6 ± 1.0  ND 86.7 ± 6.2  n = 10 n = 10 n = 8 compl.control 5.4 ± 1.2 6.2 ± 2.0 n = 6 n = 2 ND — not determined *p < 0.05 vsGr. 3 °p_(U) < 0.05 vs Gr. 3 p_(U) — non-parametrical statisticsU-criterium (Wilcoxon-Mann-Whitney) for unpaired data.

TABLE 6B SPECIFIC SUPPRESSION OF THE IMMUNE RESPONSE WITH GLYCANSCOMPLEMENT DEPENDENT CYTOTOXICITY OF MOUSE SERUM EXP. 37 TRYPAN BLUEEXCLUSION ASSAY day 30 day 60 Lymph. Lymph. Lymph. Lymph. V.L. A.B. V.L.A.B. Groups % of dead cells 1. IS + AgV.L. + Tf pool 63.7 ± 11.9  22 ±3.7 45.3 ± 12.3 17.5 ± 6.7 n = 3 n = 3 n = 3 n = 3 2. IS + AgV.L. +glycans 14.4 ± 2.2* 10.8 ± 5.1*  27.0 ± 7.1** 9.7 ± 1.1° n = 5 n = 5 n =4 n = 4 3. IS + AgV.L. + saline 63.0 ± 12.9 65.4 ± 12.5 63.9 ± 11.2 42.3± 18.3 n = 8 n = 8 n = 9 n = 4 4. non-IS + AgV.L. 100 ± 0  97.9 ± 0.7 99.8 ± 0.2  ND n = 5 n = 5 n = 5 compl. control 3.8 ± 1.3 3.8 n = 5 n =1 ND — not determined; *p < 0.01 vs Gr. 3; **p < 0.02 vs Gr. 3; °p_(U) <0.05 vs Gr. 3 p_(U) — non-parametrical statistics U-criterium(Wilcoxon-Mann-Whitney) for unpaired data

The compositions of the invention are suitable for use in many areas,some of which have already been referred to earlier. These compositionsare suitable particularly for the treatment of patients with thefollowing classes of diseases, all of which would benefit fromallogeneic or xenogeneic bone marrow transplantation:

Aplastic anemia; agranulocytosis;

Thalassemia;

Immunodeficiency diseases (AIDS, agammaglobulinemia, etc.);

Leukemias (Myeloblastic, lymphoblastic, erythroblastic, etc.)

Myelomas

Metastizing solid tumors, carcinomas, adenocarcinomas;

Genetic diseases;

Organ transplantation.

Another large group of patients can make use of the invention, e.g.,patients undergoing transplantation of organs from man or animals (pig,monkey, etc.) (Acceptance of liver, heart, kidneys, Langerhans isletswithout rejection reaction).

Whenever required the patient may be preconditioned to the immuneacceptance of an organ or tissue from another host, by prior Tf-glycansadministration and transplantation of bone marrow or bone marrow stemcells from the host which is also to provide said organ or tissue.

See also general indications supplied by Gluckman E. in its articletitled “Bilan actuel de la greffe de moelle osseuse allogénique”(General overview on the graft of allogeneic bone marrow) published inPath. Biol. 1980, 28, N° 1, 5-7. These indications are also applicablehere.

The invention finds use e.g., in allogeneic (histoincompatiblenon-HLA-matched) BMT. whereby the difficulties linked to the finding ofa donor should be circumvented to a great extent. The invention is notlimited to compositions for use in the graft of allogeneic andxenogeneic BMT only. Their use is to be contemplated in any systemaiming at facilitating the engraftment of any type of cells, tissues ororgans in any type of mammal including man.

Though the way of administering the composition of the invention and itscoupling with the steps involving depression or suppression of theendogeneous immune system in the host to be transplanted with allogeneicon xenogeneic cells should rest with the clinicians, it neverthelessremains that the abovesaid depression or suppression should normally becaused to take place prior to Tf-glycans administration andtransplantation.

Noteworthy is the fact that chemical immunosuppression of the receivinghost prior to bone marrow transplantation or transplantation of othercells tissues or organs should generally be enough. Full previousdestruction of the receiving host's own immune system does not appear asnecessary. But the systems used to induce immunosuppression in the hostmay also combine chemical immunosuppression with more or less limitedirradiation, for instance of lymphoid organs only, in order to preventlarge irradiation damage (lungs, intestine, etc.). Any cytostatic drugor immunosuppressive drug, e.g., cyclosporin, prednisolone, FK-506 maybe given alone or in combination with irradiation to condition therecipient to the transfer of foreign cells or tissues.

Neither are the uses of the compositions of the invention limited to thetransplantation of bone marrow only. They become applicable whenever atransfer into the pre-treated host of a new immunological system isrequired, e.g., for inducing rejection by the host of leukemic cells orsolid tumors. Another important use of the invention is in xenogeneic(inter-species), transplantation for example when the donor of bonemarrow or organs (e.g., liver, heart, kidney) is the pig or a primate(monkeys) and the recipient is man.

Alternatives in the time points at which the Tf-glycan pools are to beadministered to the recipient are contemplated too. They may also beadministered to the donor, prior to the transfer of his cells to therecipient. Repeated subsequent administrations of said glycan pools islikely to favor the engraftment-capacity of the bone marrow or othersorgans or tissue in the recipient, in order to reinforce the toleranceinduced towards the grafted bone marrow and organs or tissue ofallogeneic or xenogeneic origin.

Pooled Tf-glycans may also be added to bone marrow cultures, topre-incubate in vitro the donor bone marrow for variable periods (hoursor days) before its inoculation in the recipient. This procedure maychange and/or improve the engraftment capacity of the donor bone marrowand enhance induction of GvHD-free chimerism.

The compositions of the invention may be administered by any routenormally used to enhance the host non- responsiveness to the foreign(allogeneic or xenogeneic) bone marrow and/or organs. Though oral orrectal routes may be contemplated, the preferred ones remain theparenteral routes (intravenous or intramuscular injections).

Though this should not be construed in any limitative manner whatsoever,daily doses of pooled Tf-glycans to the host, after the engraftment ofbone marrow and/or organs sought to be grafted has been achieved, shouldfor instance range from about 20 micrograms to 500 micrograms, e.g.,from about 50 micrograms to about 200 micrograms per kg body weight ofthe host. Treatments of that type could last from 10 to 30 days aftersaid engraftment.

However, the treatment with the pooled Tf-glycans may also be pursueddays, weeks or months after transplantation, alone or in combinationwith immunosuppresive drugs such as e.g., cyclophosphamide, cyclosporin,FK-506, methotrexate in all those cases in which the transplantedindividual shows signs or symptoms of an ill-functioning hemopoieticsystem (anemia, leucopenia, thrombocytopenia) or of graft versus hostdisease and/or immunological deficiencies.

What is claimed is:
 1. A method for controlling an immune response to anallograft or a xenograft of foreign tissues or cells in a mammal in needof such treatment, said method comprising the steps of: (a)immunosuppressing said mammal; (b) administering pooledtransferrin-derived glycans from a mammalian species to said mammal,wherein said transferrin-derived glycans are substantially free oftransferrin polypeptide moieties; and (c) grafting said foreign cells ortissues into said mammal, wherein said foreign cells or tissues are fromthe same mammalian species as said pooled transferrin-derived glycans,thereby controlling said immune response by enchancing hostnon-responstiveness to said allograft or xenograft.
 2. The methodaccording to claim 1, wherein said pooled transferrin-derived glycansare pooled human transferrin-derived glycans.
 3. The method according toclaim 1, wherein said pooled transferrin-derived glycans contain atleast four serologically determinable antigens of HLA-A, HLA-B, HLA-C,HLA-D and HLA-DR.
 4. The method according to claim 1, wherein saidmammal is immunosuppressed with an immunosuppressive drug selected fromthe group of prednisolone, cyclophosphamide, cyclosporin, methotrexateand FK-505.
 5. The method according to claim 1, wherein said foreigncells are bone marrow cells.
 6. The method according to claim 1, whereinsaid foreign cells are leukocyte cells.
 7. The method according to claim1, wherein said foreign cells are peripheral blood progenitor cells. 8.A method for controlling an immune response to an allograft or axenograft of foreign tissues or cells in a mammal in need of suchtreatment, said method comprising the steps of: (a) immunosuppressingsaid mammal; (b) administering transferrin-derived glycans to saidmammal, wherein said trannsferrrin-derived glycans are substantiallyfree of transferrin polypeptlide moleties; and (c) grafting said foreigncells or tissues into said mammal, wherein said foreign cells or tissuesoriginate from the same mammalian donor from which saidtransferrin-derived glycans are obtained, thereby controlling saidimmune response by enhancing host non-responstiveness to said allograftor xenograft.